bioRxiv preprint doi: https://doi.org/10.1101/2020.09.22.306811; this version posted September 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
First high-quality reference genome of Amphicarpaea edgeworthii
Tingting Song1+, Mengyan Zhou2+, Yuying Yuan1, Jinqiu Yu 1, Hua Cai1,
Jiawei Li1, Yajun Chen1, Yan Bai2, Gang Zhou2 and Guowen Cui1*
1 Northeast Agricultural University, 150030, Harbin, China
2 Novogene Bioinformatics Institute, 100083 Beijing, China
+ These authors contributed equally
* Corresponding author:
Guowen Cui, Northeast Agricultural University, Harbin, No. 600 Changjiang Road,
Harbin, 150030, China.
Email: [email protected]
1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.22.306811; this version posted September 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Abstract
Amphicarpaea edgeworthii, an annual twining herb, is a widely distributed species
and an ideal model for studying complex flowering types and evolutionary
mechanisms of species. Herein, we generated a high-quality assembly of A.
edgeworthii by using a combination of PacBio, 10× Genomics libraries, and Hi-C
mapping technologies. The final 11 chromosome-level scaffolds covered 90.61% of
the estimated genome (343.78 Mb), which is the first chromosome-scale assembled
genome of an amphicarpic plant. These data will be beneficial for the discovery of
genes that control major agronomic traits, spur genetic improvement of and functional
genetic studies in legumes, and supply comparative genetic resources for other
amphicarpic plants.
2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.22.306811; this version posted September 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Introduction
In nature, the distribution of key resources required for plant growth is often
uneven. Plants growing in unstable habitats, with limited supplies of mineral nutrients,
water, or light, frequent soil interferences, and large environmental fluctuations,
undergo adaptive evolution to improve their survival(Jackson and Caldwell, 1993;
Pearcy and Caldwell, 1994). Some plant species that bear 2 or more heteromorphic
flowers also bear heteromorphic fruits (seeds). Amphicarpy is a phenomenon in which
a plant produces both aerial and subterranean flowers and simultaneously bears both
aerial and subterranean fruits (seeds) on aerial- and subterranean- stems, respectively
(Cheplick, 1987; Koontz et al., 2017; Schnee and Waller, 1986). This phenomenon is
observed in at least 67 herbaceous species (31 in Fabaceae) in 39 genera and 13
families of angiosperms, as reported by Zhang et al(Zhang et al., 2020a). Amphicarpy
is an important part of plant adaptive evolution, in which angiosperms generally
display a special type of fruiting pattern and different fruit (seed) types also exhibit
various dormancy and morphological features. This type of fruiting mode is crucial
for the ecological adaptation of plants that have evolved through natural selection
because it reduces competition among siblings within the population, maintains and
increases the population size in situ, and increases the adaptability and evolutionary
plasticity of the species(Hidalgo et al., 2016; Sadeh et al., 2009).
Amphicarpaea edgeworthii, an annual twining herb, belongs to the Fabaceae
family, which is a large and economically valuable family of flowering plants(Zhang
et al., 2017; Zhang et al., 2006). In this plant species, 3 types of flowers (fruits) can
3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.22.306811; this version posted September 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
grow in the same plant, and aerial chasmogamous flowers are produced only during
summer( Zhang et al., 2006; Zhang et al., 2005). This species may offer an attractive
model for examining gene regulatory networks that control chasmogamous and
cleistogamous flowering in plants. However, the mechanism of flower development in
amphicarpic plants, particularly in legumes, is sparsely known. The present study was
an attempt to enhance our understanding on the reproductive biology and the ultimate
evolutionary mechanism in amphicarpic plants. We performed whole-genome
sequencing of A. edgeworthii, which is the first plant in the legume family and also
the first amphicarpic species subjected to sequencing. This reference genome
represents a precious foundation for further understanding on agronomics and
molecular breeding in A. edgeworthii.
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Result
A. edgeworthii has a diploid genome (2n = 2x = 22) (Supplementary Figure 1).
We estimated the genome size of A. edgeworthii to be 360.91 Mb using
17-mer(Supplementary Table 1 and Figure 2). We sequenced the genome of A.
edgeworthii by using a combination of PacBio, Illumina, and 10× Genomics libraries
that resulted in the generation of a 343.78-Mbp genome (contig N50 length = 1.44 Mb,
scaffold N50 length = 2.4 Mb; Table 1 and Supplementary Table 2, 3 and 4). Finally,
we assembled a chromosome-level genome by using Hi-C technology. We used a
total of 5.27 million reads from Hi-C libraries and mapped approximately 90.61% of
the assembled sequences to 11 pseudochromosomes, with the longest scaffold length
of 32.05 Mb (Table 1 and Figure 1). Results indicate that the A. edgeworthii genome
was adequately covered by the assembly.
We evaluated the completeness of the genome assembly by mapping the Illumina
paired-end reads to our assembly through Burrows–Wheeler Alignor (BWA) (Li and
Durbin, 2009) with 98.70 % of mapping rate and 94.04 % of coverage
(Supplementary Table 5). Then, we used both Core Eukaryotic Gene Mapping
Approach (CEGMA) (Parra et al., 2007) and Benchmarking Universal Single-Copy
Orthologs (BUSCO) (Simão et al., 2015) to access the integrity of the assembly. In
the CEGMA assessment, 238 (95.97%) of 248 core eukaryotic genes were assembled
(Supplementary Table 6). Furthermore, 93.4% complete single-copy BUSCOs were
detected, which indicates that the assembly was complete (Supplementary Table 7).
Overall, the results indicate that a high-quality assembly was generated.
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Repeat sequences comprise 51.28% of the assembled genome, with transposable
elements (TEs) being the major component (Supplementary Table 8). Among the TEs,
long terminal repeats (LTRs) were the major component (29.32%) (Supplementary
Table 9). We combined de novo prediction, homology search, and mRNA-seq
assisted prediction to predict genes in the A. edgeworthii genome, and we obtained
28,372 protein-coding genes (97.2% were annotated) (Supplementary Tables 10 and
11). Additionally, we identified 2,260 non-coding RNAs, including 471 miRNAs, 701
transfer RNAs, 266 ribosomal RNAs, and 822 small nuclear RNAs (Supplementary
Table 12).
Table 1 Statistics of the A. edgeworthii genome assembly
Total assembly size (Mb) 343.78
Total number of contigs 1,475
Total number of scaffolds 1,082
Contig N50 length (Mb) 1.44
Maximum contig length (Mb) 7.65
Maximum scaffold length (Mb) 32.05
Scaffold N50 length (Mb) 28.47
Scaffold N90 length (Mb) 23.07
GC content (%) 32.04
Gene number 28,372
Repeat content (%) 51.28
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Discussion
In this study, we constructed a high-quality chromosome-level genome assembly
for A. edgeworthii by combining the long-read sequences from PacBio with highly
accurate short reads from Illumina sequencing and using Hi-C technology for
super-scaffolding. The assembly of A. edgeworthii adds to the growing genomic
information on the Fabaceae family. As the first species in the Amphicarpaea genera
of the Fabaceae family to be sequenced, A. edgeworthii exhibits a range of specific
biological features, such as 3 types of flowers and 3 types of fruit (seed)
simultaneously in the same plant. It has maintained an essential evolutionary position
in the tree of life(Goodwillie et al., 2005; Silvertown et al., 2001). According to our
analysis, these genomic data will serve as valuable resources for future genomic
studies on amphicarpic plants and molecular breeding of soybean.
To date, no genomic data for the amphicarpic plants are available. Therefore, the
sequencing of the entire A. edgeworthii genome will facilitate future investigations on
the phylogenetic relationships of the flowering (seed) plants. The accessibility of the
A. edgeworthii genome sequence makes feasible the investigation of deep
phylogenetic questions on angiosperms and the determination of genome evolution
signatures and the genetic basis of interesting traits.
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Method
Plant material and genome survey
For genome sequencing, we collected fresh young leaves of A. edgeworthii
species distributed in Heilongjiang Province (45.80°N, 126.53°E), China. The
karyotype analysis of the plant species revealed a karyotype of 2n = 2x = 22, with
uniform and small chromosomes(Wolny et al., 2013) (Supplementary Figure 1).
We extracted DNA from the fresh leaves of A. edgeworthii by using a DNAsecure
Plant Kit(TIANGEN, Biotech, China) and then purified and concentrated,
high-quality DNA was broken into random fragments, and Illumina paired-end library
with 350-bp size was constructed and was sequenced using a Illumina HiSeq X-ten
platform.
To estimate the A. edgeworthii genome size, high-quality short-insert reads
(350-bp size) were used to extract the 17-k-mer sequences by using sliding windows.
The frequency of each 17-k-mer was calculated and is presented in Supplementary
Figure 2. Genome size was calculated by using the following formula:
Genome size = total k-mer numbers/k-mer depth
The revised genome size was calculated after excluding the erroneous k-mers
(Supplementary Tables 1).
Library construction, genome sequencing, assembly, and evaluation
To construct long-insert libraries, the SMRTbell libraries were constructed by
following the standard protocol as per the manufacturer’s instructions (PacBio
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Biosciences). Genomic DNA was broken into fragments of 15 kb–40 kb in size and
the large fragments were enriched and enzymatically repaired and converted into
SMRTbell libraries. The SMRTbell libraries were sequenced using a PacBio Sequel
platform.
The linked read sequencing libraries of 10× Genomics GemCode
platform(Weisenfeld et al., 2017) were sequenced with 350-bp size by using an
Illumina HiSeq X-ten platform.
Fresh leaves were plucked from the plant and chromatin in the samples were
cross-linked to DNA and fixed. A chromatin interaction mapping (Hi-C) library with
350-bp size was constructed for sequencing using Illumina HiSeq X-ten.
We used FALCON software(Chin et al., 2016) for de novo assembly of PacBio
SMRT reads (Supplementary Tables 2 and 3). The longest coverage of subreads were
selected as seeds for assembly after pairwise comparisons of all reads for error
correction with default parameters. The error-corrected SMRT reads were aligned to
each other to construct string graphs. After initial assembly, the produced contigs
were polished using Quiver(Chin et al., 2013) with default parameters. The first round
of error correction was performed using Illumina paired-end reads by Pilon(Walker et
al., 2014). Subsequently, the scaffolding was performed using 10× Gscaff v2.1 with
10× genomics data, and the genome was upgraded by PBjelly(English et al., 2012).
The second round of error correction was performed using Illumina paired-end reads
by Pilon(Walker et al., 2014). The Hi-C data were mapped to the original scaffold
genome by using BWA v0.7.7(Li and Durbin, 2009), and only the reads with unique
9 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.22.306811; this version posted September 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
alignment positions were extracted to construct a chromosome-scale assembly by
using the Ligating Adjacent Chromatin Enables Scaffolding In Situ(LACHESIS)
tool(Burton et al., 2013) (Supplementary Table 3).
Genome annotation
We used RepeatModeler, RepeatScout(Tarailo and Chen, 2009), Piler(Edgar and
Myers, 2005), and LTR_FINDER(Xu and Wang, 2007) to develop a de novo
transposable element library. RepeatMasker(Tarailo and Chen, 2009) was used for
DNA-level identification in the Repbase and de novo transposable element libraries.
Tandem repeats were ascertained in the genome by using Tandem Repeats
Finder(Benson, 1999). RepeatProteinMask(Tarailo and Chen, 2009) was used to
conduct WU-BLASTX searches against the transposable element protein database.
Overlapping TEs belonging to the same type of repeats were integrated
(Supplementary Tables 8 and 9).
To predict protein-coding genes in the A. edgeworthii genome, we used
homolog-based prediction (using A. duranensis, C. arietinum, G. max, M. truncatula,
P. vulgaris, and T. pratense gene sets), de novo prediction (using Augustus
v.3.0.2(Stanke and Morgenstern, 2005), Genescan v.1.0(Aggarwal and Ramaswamy,
2002), GeneID(Parra et al., 2000), GlimmerHMM v.3.0.2(Majoros et al., 2004), and
SNAP(Korf, 2004) programs) and transcriptome-based prediction (using 5 tissue
RNA sequencing data). A weighted and non-redundant gene set was generated using
EVidenceModeler (EVM) (Brian et al., 2008), which merged all the genes models
that were predicted using the aforementioned approaches. Along with the transcript
10 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.22.306811; this version posted September 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
assembly, the Program to Assemble Spliced Elements(Haas et al., 2003) was used to
adjust the gene models generated using EVM (Supplementary Table 10).
Functional annotation of protein-coding genes was evaluated using BLASTP
(E-value ≤ 1E-05) against 2 integrated protein sequence databases, SwissProt(Bairoch
and Apweiler, 2000) and NCBI non-redundant protein database. Protein domains
were annotated by searching InterPro v.32.0, which included Pfam, PRINTS,
PROSITE, ProDom, and SMART databases, by using InterProScan v.4.8(Mulder and
Apweiler, 2007). GO(Ashburner et al., 2000) terms for each gene were obtained from
the corresponding InterPro descriptions. The pathways in which the gene might be
involved were assigned using BLAST searches against the KEGG database(Kanehisa
and Goto, 2000), with an E-value cutoff of 1E-05 (Supplementary Table 11).
The tRNA genes were predicted using tRNAscan-SE software(Lowe and Eddy,
1997). The miRNA and snRNA fragments were identified using INFERNAL
software(Nawrocki et al., 2009) against the Rfam(Griffiths et al., 2005) database. The
rRNA were identified using BLASTN searches (E-value ≤ 1E-10) against the plant
rRNA database (Supplementary Table 12).
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Acknowledgement
This research work was funded by the National Key R&D Project of China
(2016YFC0500607). We are also grateful to Ms. Mei Zhang, Mr. Bo Zhao and Ms.
Junyan Li (Novogene Bioinformatics Institute, Beijing China) for providing helps in
the project.
Author contributions
G.W.C.and T.T.S. conceived and designed the project and the strategy; T.T.S,
Y.Y.Y, J.Q.Y and J.W.L. collected and cultured the plant material, DNA/RNA
preparation, library construction and sequencing; T.T.S and M.Y.Z worked on
genome assembly and annotation, comparative and population genomic analyses, and
transcriptome sequencing and analysis; G.W.C., T.T.S, M.Y.Z, H.C., Y.J.C, Y.B, and
G.Z contributed substantially to revisions. All authors commented on the manuscript.
Conflict of Interest
The authors declare that they have no conflict of interest.
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Figure 1. Characteristics of the A. edgeworthii genome: a. Chromosome length; b.
Gene density per chromosome; c. Repeat density; d. LTR_Copia density; e.
LTR_Gypsy density; f. SNP density in 5 populations; g. Distribution of GC content; h.
Intra-genome collinear blocks connected. All statistics are computed for windows of
200 kb.
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